Endophytic microbial strains as biocatalysts isolated from fresh plant samples, compositions, and methods of use thereof to enhance the growth and/or yield of a plant in the presence of reduced synthetic phosphate fertilizers are provided. Endophytic microbial strains serve as biocatalysts to solubilize mineral-P and mineralize organic-P otherwise unavailable to plants for their nutritional phosphate needs. Thus defined, biocatalysts, will serve to replace synthetic phosphate fertilizers. Also provided are materials and methods for inoculating plants with these biocatalysts at carefully selected inoculum densities to reliably reduce the amount of synthetic phosphate fertilizer by 50% thus accomplishing obtaining optimal yields in technically and cost-effective manner.
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1. A synthetic combination comprising a purified bacterial population in association with a seed of an agricultural plant, wherein the purified bacterial population comprises a plant bacterial endophyte that is heterologous to the seed, having all the identifying characteristics, of NRRL B 67827, and is capable of producing organic acids, acid phosphatase, and alkaline phosphatase enzymes, to solubilize soil phosphorus at acid and alkaline soil pHs and wherein the plant bacterial endophyte is present in the synthetic combination at a concentration of at least 105 CFU/seed on the surface of a seed or inside the seed and in an amount effective to reduce phosphorus fertilizer requirement for optimal plant growth and yield in organic and conventional agriculture compared to a reference agricultural plant grown under the same conditions with no reduction in phosphorus fertilizer.
2. The synthetic combination of
3. The synthetic combination of
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5. The synthetic combination of
6. The synthetic combination of
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8. The synthetic combination of
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The present invention relates to the field of sustainable agriculture. Specifically, the disclosure provides novel purified bacterial populations comprising plant isolated endophytes and synthetic combinations of seeds with heterologously plant derived endophytes to provide a specific benefit that of optimal production of crop plants in the absence of synthetic phosphate fertilizers or in the presence of synthetic phosphate fertilizers applied at the rate of 30-50% less than the recommended phosphorus fertilizer application rate. In particular, the synthetic compositions and methods disclosed herein are useful for enhancing plant growth in the complete absence of synthetic phosphate fertilizers or in their much reduced presence.
The United States is, by far, the largest producer of corn in the world, producing as much as 35% of world's corn, 33% of world's soybean, more than 65% of world's sorghum and is the biggest wheat exporter (Ag 101, US EPA).
Although US currently leads the world in corn production, the portion of agricultural revenue returned to farmers decreased from 37% to 19% from 1950 to 2002 (Atwell et al., 20111). In addition, input costs increased sevenfold and the real price of corn adjusted for inflation decreased fivefold (Duffy, 2006).
One major contributor to the input costs is the cost of fertilizer and pesticides because US farming is fertilizer and pesticide intensive.
The cost of phosphate fertilizer has risen steadily from the year, 2000 doubling in the year 2007(Oleyzk et al., 2007).
Phosphorus fertilizer manufactured from phosphorus rock is mined in the US (Florida), China, Morocco, and Russia. In year 2006 alone, 142 million tons was mined worldwide (Oleyzk et al., 2007), yet, at the current world-wide rate of application of fertilizers, the readily available sources of high grade phosphate rocks will be depleted within the next 60 to 90 years (Runge-Metzger, 1995).
The situation is critical because the supply of phosphate rock in Florida may be exhausted as early as year 2040 according to the Institute of Phosphate Research (Oleyzk et al., 2007).
Phosphate fertilizers have been critical to crop growth because phosphorus deficiency often limits plant growth (Schachtman, et al., 1998; Vance, et al., 2003; Raghothama, and Kartikeyan, 2005) although this essential plant macronutrient is critically important for improving soil fertility in both tropical and temperate regions (Von Uexküll & Mutert, 1995).
The practice of applying chemical (inorganic) P fertilizers to alleviate P deficiency is inefficient for both logistical and economic reasons however, because 75-90% of soluble P from fertilizers rapidly becomes immobilized as Fe-, Al-, or Ca—PO4 phases in soils (Gyaneshwar et al., 2002).
Thus effective means for utilizing residual accumulated insoluble P in soils and a means for increasing efficiency of applied synthetic P fertilizers are critically needed.
This takes on particular significance when we note that global food production needs to increase by 50% in the next 20 years to sustain the increasing world population and prosperity.
Although theoretical estimates have suggested that the accumulated P in agricultural soils is sufficient to sustain maximum crop yields worldwide for about 100 years (Gyaneshwar et al., 2002), most soils are deficient in bioavailable P.
Therefore, sustainable alternatives to improving P bioavailability are needed for maintaining U.S.'s edge in crop production and agricultural productivity in general.
The proposed embodiment will address the current need for sustainable and cost efficient P management in soils by developing a biocatalyst that makes mineral soil-P, and organic-P available for plant needs thus reducing or eliminating the need for the application of synthetic P fertilizers by a combination of utilization of already available soil-P and by increasing the efficiency of applied synthetic-P fertilizer.
The proposed embodiment will have the additional benefit of improving surface and ground water quality because application of manures and fertilizers has resulted in increased transfer of soil P (solid associated) to solution and eventually, via erosion and runoff, to surface waters where it plays a key role in eutrophication and impairment of affected waters as a resource for drinking, recreation and industry.
The total soil P content typically varies between 500 and 2000 mg kg−1 (Vance et al., 2003).
Of this, typically 30-50% of the total insoluble P is present as organic P (Po) mainly as inositol phosphate and the remaining is found as Fe-, Al- or Ca-associated mineral phosphate (Pi); phosphate dissolved in soil solution ranges between only 0.1 and 10 μM (Bielski, 1973; Ozanne, 1980; Raghothana, 1999; Frossard et al., 2000).
Because plants can only take up phosphorus as dissolved HxPO4y− ions, and for optimal crop growth 0.5-0.7 mM dissolved P is needed in the soil solution, most soils are P deficient and crop growth is often limited by P bioavailability.
Application of chemical fertilizers i.e., phosphate salts is used to supplement the limited pools of dissolved P; however, because of high affinity of P binding to Fe-oxyhydroxide, Al-oxyhydoxide minerals and precipitation as calcium-phosphate phases, dissolved P is quickly converted to insoluble P.
The main goal of the preferred embodiment then is to increase the efficacy of chemical fertilizers and to use the already existing substantial reserves of insoluble soil-P, thereby reducing the application of chemical fertilizers.
Toward this goal, we enhance dominant natural mechanisms of insoluble soil-P bioavailability.
Two main mechanisms of making Pi and Po bioavailable for plant's nutritional needs involves secretion of organic acids to solubilize Pi and secretion of phosphatase enzymes for mineralization of Po. These organic acids and phosphatase enzymes are secreted both by soil microorganisms (bacteria and fungi) and to a smaller extent by plant roots in response to P deficiency (Raghothana and Karthikeyan, 2005; Martinez, 1967).
Although both bacteria, and fungi are ubiquitous in soils, Pi solubilizing and Po mineralizing bacteria (phosphobacteria) generally outnumber their fungal counterparts by 2-150 fold (Hilda and Fraga, 1999); the P solubilization potential of phosphobacteria can therefore be enhanced to serve as an effective biocatalyst in making insoluble ‘fixed’ P plant available in an eco-friendly, reliable and sustainable manner.
Phosphobacteria have both epiphytic and endophytic modes of association with the host plant (corn, soybean, wheat, and sorghum, and other crops by extension) and the mode of association can affect the efficacy of phosphobacteria.
Colonizing the plant root epiphytically is difficult because the inoculant has to compete with the native soil bacteria (Kozyrovska et al., 1996).
Phosphobacteria with endophytic relationship with host plant reside within apoplastic spaces inside the host plant thus keeping them away from the natural biocenosis giving them a significant edge in competing with the soil bacteria (Kozyrovska et al., 1996; Sturz et al., 2000).
Because, endophytes live within the plant, they can recover more easily from stress situation; they may also form beneficial host-endophyte allelopathies thus protecting the plant from superinfection by soil bacteria (Kozyrovska et al., 1996; Sturz et al., 2000).
The proposed embodiment pertains to the development of biofertilizer consisting of endophytic phosphobacteria (BioCat-P) inoculated crop seeds as an environmentally sustainable, and cost-effective alternative to synthetic fertilizers and/or applied in combination with the synthetic-P fertilizers as a means of increasing the efficacy of synthetic fertilizers. The present invention relates to the field of sustainable agriculture. Specifically, the disclosure provides microbial compositions and methods useful for the optimal production of crop plants in the absence of synthetic phosphate fertilizers or in the presence of synthetic phosphate fertilizers applied at the rate of 30-50% less than needed. In particular, the compositions and methods disclosed herein are useful for enhancing plant growth in the complete absence of synthetic phosphate fertilizers or in their much reduced presence.
As part of our invention, we isolated and purified a number of endophytic bacteria from corn, sorghum and sugarcane plant parts. All the isolated and purified bacterial strains were screened quantitatively for their ability to function as endophytic phosphobacteria. This was accomplished by screening for production of organic acids for solubilizing inorganic/mineral phosphorus; and for the production of acid and alkaline phosphatase enzymes for conversion of organic phosphorus to inorganic phosphorus. In addition, phosphobacteria were tested for heterologous inoculation of corn and sorghum seeds.
We were able to purify 12 bacterial strains of endophytic phosphobacteria from corn, sorghum, and sugarcane plants of which only one strain T6 (BMS-201, NRRL accession #67827) showed production of all three extracellular enzymes for phosphorus solubilization that include organic acids, acid phosphatase, and alkaline phosphatase enzymes. This strain has been deposited as BMS-201 with the NRRL and has the accession number B-67827.
The preferred embodiment comprising synthetic combination of the purified bacterial strain T6 (BMS 201; NRRL accession number B-67827) when tested for its heterologous inoculation of corn and sorghum seeds showed excellent inoculation ability without harming the plant. The preferred embodiment comprising synthetic combination of the purified bacterial strain T6 heterologously inoculated corn and sorghum seeds were evaluated in crop systems in hydroponic, simulated soil systems, and real soils.
The preferred embodiment has included isolation of Pi solubilizing and acid- and alkaline-phosphatase producing endophytic bacteria from plant samples of corn, sorghum, and sugarcane.
Further, the preferred embodiment has involved determining inoculation efficacy of the isolated endophytic bacteria by determining inoculum density in root and shoot of corn and root and shoot of sorghum.
Further, the preferred embodiment has involved using phosphate deficiency symptoms, root/shoot ratio, and chlorophyll content measurements in demonstrating that corn and sorghum seeds inoculated with selected Pi solubilizing endophytic bacteria grown with insoluble mineral-P species present in soils such as rock-phosphate and iron-phosphate performed as well or better than non-inoculated control seeds grown with full strength Hoagland solution containing dissolved P.
In
In
‘Soil phosphorus’ is defined as the sum total of soil-mineral phosphorus and soil-organic phosphorus.
An ‘agricultural plant’ can be a monocotyledonous or a dicotyledonous plant typically used in agriculture.
A ‘host agricultural plant’ is a monocotyledonous or a dicotyledonous plant typically used in agriculture
A ‘reference plant’ is a monocotyledonous or a dicotyledonous plant typically used in agriculture that has not been synthetically inoculated by an ‘endophytic phosphobacteria’ and serves as control against which the performance of the ‘host agricultural plant’ may be compared to draw conclusions about the success of the invention.
Fresh samples of corn, sorghum, and sugarcane plants were acquired and washed in tap water. The roots and shoots from each plant were separated and chopped. They were then surface sterilized to eliminate any epiphytic bacteria and to facilitate isolation of only endophytic bacteria. The samples were then ground to isolate endophytic bacteria. Endophytic bacteria isolated from corn plants included T4, T6 (BMS-201, NRRL accession #67827), and C8. Endophytic bacteria isolated from sorghum plants included J-1, J-2/1, J-2/2, J-3/1, J-3/2, J-3/3, and J-4. Endophytic bacteria isolated from sugarcane plants included S-1/1, S-1/2, S-5, S-7, and S-8.
Sperber's media for screening Pi solubilizing endophytic bacteria: The basal Sperber (1958) medium was used and contained glucose 10.0 g/l, yeast extract 0.5 g/l, CaCl2) 0.1 g/l, MgSO4.7H2O 0.25 g/l and agar 15.0 g/l. The medium was supplemented with 2.5 g/L of Ca3(PO4)2 (TCP-tricalcium phosphate) as P source to appraise the ability of the strains to mobilize inorganic P sources. The pH of the medium was adjusted to 7.2 before autoclaving. The media were distributed in 9 cm diameter Petri plates and marked in four equal parts after solidification. Using the drop plate method, each part was inoculated with innocula. All tests were performed with four replications. Inoculated plates were incubated in dark at 27 degree C. and the diameter of clear zone (halo) surrounding the bacterial growth as well as the diameter of colony were measured after 10, 20 and 30 days.
TABLE 1
Results of Sperber's PO4 solubilizing Assay
Endophytic bacteria isolated
from corn (C, T), surgarcane
Phosphate solubilizing
(S) and sorghum (J)
activity (zone in cm)
C-8
Very low (0.1 cm)
T-4
Medium (1 cm)
T-6
Medium (0.6 cm)
J-2/2
Very low (0.2 cm)
J-3/2
Very low (0.1 cm)
J-3/1
Very low (0.3 cm)
J-4
Medium (1 cm)
S-1/1
Very low (0.1 cm)
S-1/2
Very low (0.3 cm)
S-5
Very high (4 cm)
S-7
Very high (5 cm)
S-8
High (3 cm)
Screening for Acid Phosphatase Producing Endophytic Bacteria:
The isolated strains were grown in 50 ml of liquid medium (0.1% Ca-phytate; 1.5% glucose; 0.2% NH4NO3; 0.05% KCl; 0.05% MgSO4.7H2O; 0.03% MnSO4.4H2O; 0.03% FeSO4.7H2O, pH 5.5) in 500-ml flask and incubated at 28 degree C. for 48 hours on reciprocal shaker (200 rpm). The cells were collected from 1 ml of culture by centrifugation at 5000×g for 10 minutes in cool room (40C) and re-suspended in acetate buffer (0.2 M, pH 5.5). The reaction mixture was prepared. It consisted of 0.8 ml acetate buffer (0.2 M, pH 5.5) containing 1 mM Na-phytate and 0.2 ml of cell suspension. After incubation for 30 minutes at 37 degree C., the reaction was stopped by adding 1 ml of trichloroacetic acid. One ml aliquot was analyzed for inorganic phosphate liberated using the colorimetric procedure. One unit of enzyme activity was defined as the amount of enzyme liberating 1 n mol of inorganic phosphate per minute.
TABLE 2
Results of acid phosphatase assay
Endophytic bacteria isolated
Concentration of acid
from corn (C, T), surgarcane
phosphatase produced
(S) and sorghum (J)
(mg/l)
J-1
0.60
S-5
0.60
C-8
0.58
J-4
0.57
S-7
0.62
J-2/2
0.52
T6 (BMS-201, NRRL
0.60
accession #67827)
J3/1
0.62
T4
0.61
S-1/2
0.60
J-2/1
0.59
Screening for alkaline phosphatase producing endophytic bacteria: The endophytic bacteria were grown in blood agar for 24 h. One colony was transferred and incubated at 37 degree C. in 2.75 ml of propanediol buffer (0.2 mol/liter, pH 7.5) containing 2 mg of 5-bromo-4-chloro-3-indolyl phosphate previously dissolved in 0.25 ml of N,N-dimethyl formamide. 0.2 ml of MgCl2 (5 mmol/liter) was added as an activator. Alkaline phosphatase production was examined every 30 minutes for 4 h by looking for a blue-green indigo precipitate development on the bacterial growth causing the entire solution to become blue.
TABLE 3
Results of alkaline phosphatase assay
Endophytic bacteria isolated
from corn (C, T), surgarcane
Change of color to
(S) and sorghum (J)
indigo blue-green
C-8
Negative
T-4
Negative
T-6
Positive
J-1
Negative
J-2/1
Negative
J-2/2
Negative
J-3/2
Negative
J-3/1
Negative
J-3/3
Negative
J-4
Negative
S-1/1
Negative
S-1/2
Negative
S-5
Negative
S-7
Negative
S-8
Negative
The inoculum for endophytic bacteria was grown under controlled conditions for 48 hrs to inoculum density of 108 to 1010 cfu/ml. The inoculum was centrifuged and suspended in sterile PBS to a concentration of 108 cfu/ml. The seeds were surface sterilized with 95% ethanol for 2 min and 2.5% sodium hypochlorite for 20-30 min followed by washing seven times in sterile water. Surface sterilized seeds were soaked in sterile PBS containing endophytic bacteria and placed in a temperature controlled incubator shaker at 25 degree C. for exactly 30 minutes. The inoculated seeds were washed with 70% alcohol for 2 minutes and with 2% sodium hypochlorite followed by washing with sterile water 5 times. The surface sterilized seeds were placed in sterile petriplates containing 0.7% of water agar, 5-10 seeds per plate. The seed containing plates were transferred to growth chamber set at 30 degree C. and left for 48 hours to germinate. Well germinated seeds with shoot and roots were separated and surface sterilize with 95% of ethanol for 5 min and 20 min with 4% sodium hypochlorite followed by 4-5 times sterile water rinse. The water rinsed root and shoot parts were transferred to PBS containing solution and ground to rapture the tissue. 1 ml of ground tissue was diluted in 9 ml of sterile water serial dilutions were continued to obtain 100 and 1000 fold dilution and spread on nutrient agar plates. After growth the colonies were counted and tabulated. Non-inoculated seeds served as negative controls.
TABLE 4
Inoculation efficacy of endophytic bacteria in corn and sorghum
cfu/ml (calculated using
Sample description
1000 fold dilution)
Corn-root inoculated with T6
2.1 × 106
(BMS-201, NRRL accession
#67827)
Corn-shoot inoculated with T6
1.7 × 105
(BMS-201, NRRL accession
#67827)
Sorghum-root inoculated with T6
2.6 × 106
(BMS-201, NRRL accession
#67827)
Sorghum-shoot inoculated with T6
3.4 × 105
(BMS-201, NRRL accession
#67827)
Un-inoculated corn and sorghum seeds showed zero inoculum density in roots and shoots
Standard Hoagland solutions (hydroponic nutrient solutions) were prepared and contained Ca(NO3)2.4H2O, NH4NO3, KCl, KNO3, Mg(NO3)2.6H2O, KH2PO4, Fe(N3)3.9H2O, Na HEDTA, MnCl2.4H2O, H3BO3, ZnSO4.7H2O, CuSO4.5H2O, and Na2MoO4.2H2O. The young corn seedlings cannot tolerate full strength Hoagland solution. Hence ½ strength Hoagland solution was used from VE to V1 vegetative stage. The plants were grown until V3 vegetative growth stage because phosphate deficiency symptoms can be observed during V1 to V3 growth stage. Phosphate deficient Hoagland solution was prepared by eliminating KH2PO4.
The viable inoculated seeds were placed in a muslin cloth and washed with running tap water and dried by placing on autoclaved tissue paper. The seeds were surface sterilized in laminar flow hood with 70% alcohol for 30 minutes and then by washing with 10% sodium hypochlorite solution for 20 minutes. After surface sterilization, the seeds were washed with sterile water, excess water removed by blotting with autoclaved tissue paper, and then air dried under laminar flow. Five to ten surface sterilized and dried seeds were then placed in sterile petriplates containing water soaked blotting paper and transferred to growth chamber maintained at 25-27 degree C. and left for 48 hours to germinate. After seeds germinated, they were transferred to hydroponic reactors. Hydroponic systems were maintained under greenhouse conditions at 23-24 degree C., 70% relative humidity, and 12 hours photo-period. Seeds were first grown in full strength Hoagland solution and then transplanted into hydroponic reactors for all the treatments and controls. Five replications were used for all treatments and controls.
Hydroponic reactors constituted of root permeable plastic buckets. Air pump with air controllers were used to provide aeration to the plants in hydroponic systems and all systems were maintained under greenhouse conditions at a temperature of 24±2 degree C. and relative humidity of 70±3%. After control plants showed phosphate deficiency symptoms, the plants were removed from hydroponic reactors and washed under running tap water to completely remove Hoagland solution. The plant was dried with blotting paper while taking care to not damage the roots. The roots were separated from the shoots by using sharp scissors and were weighed to record fresh weight of samples. The root and shoot samples were also dried in the dry air oven at 40 degree C. for 2 days and their dry weight was recorded. The root:shoot ratio of individual plants was calculated and recorded.
Reduced leaf area and degradation of chlorophyll in leaves is also symptomatic of phosphate deficiency so we also measure chlorophyll a and chlorophyll b in plants. A single leaf per plant was used for obtaining 10 leaf discs of 1 cm each and weighed. Five of these leaf discs were placed per tube containing 5 ml of 1:1 ratio of DMSO:acetone and the tubes were placed in the dark overnight to allow chlorophyll leaching. After chlorophyll leaching the solution turns green and the concentration of chlorophyll in leaves is calculated by measuring absorbance at 645, and 663 nm. The total chlorophyll is estimated using the following formulas:
Chlorophyll II a (g/l)=0.0127 A663−0.00269 A645
Chlorophyll II b (g/l)=0.0029 A663−0.00468 A645
Total Chlorophyll (g/l)=0.0202 A663+0.00802 A645
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